The aim of the proposed project is to study the structure and function of the DNA-protein covalent complex in Bacillus subtilis bacteriophage phi29. We will study: a) the mechanism of phi29 DNA replication and the role of protein p3 in this process. Phi29 DNA replicative intermediates will be analyzed by electron microscopy and the origin and termination of replication will be determined. The function of gene products involved in phi29 DNA replication will be studied by shift-up experiments in bacteria infected with ts mutants and analysis of the labelled DNA by alkaline sucrose gradients. The involvement of the parental protein p3 in the initiation of replication will be studied by infection of B. subtilis, grown in a heavy medium, with light sus3-mutants; b) the structure of the DNA-protein complex. The nature of the linkage between protein p3 and DNA will be determined after nuclease and protease treatments. The nucleotide sequences at the phi29 DNA termini will be studied by using the Maxam and Gilbert technique. The location of the DNA-p3 linkage in the protein, the amino acid sequence around the DNA-protein bond and the DNA sequence coding for protein p3 will be determined; c) the in vitro formation of the DNA-protein complex. Protein p3 will be purified in a native state and it will be incubated with protein-free phi29 DNA or with the different deoxyribo- or ribonucleoside triphosphates in the presence of DNA-protein p3 complex. The existence of a nucleotide attached to protein p3 isolated from infected cells will also be studied; d) the possible role of protein p3 in DNA encapsulation. New ts mutants in cistron 3, able to replicate the viral DNA but not to produce virus progeny, will be sought. The in vitro packaging of phi29 DNA-protein p3 complex or of protein-free DNA will be also studied.